Project Summary Olfaction is essential for detecting volatile molecules in the environment, is crucial for the survival of many vertebrate species, and contributes significantly to quality of life in humans. In mammals, odors are detected by neurons in the main olfactory system using a large family odorant receptor (OR) genes. These genes show an unusual form of gene regulation?each olfactory neuron expresses a single allele of a single OR gene. The mechanism underlying this random monoallelic (or ?monogenic?) expression is not understood, but likely involves the interaction of proximal and distal cis-acting elements, as well as epigenetic mechanisms. Mammals also retain a smaller family of main olfactory receptors, the Trace Amine Associated Receptors (TAARs). The TAARs are also subject to monogenic expression. However, recent data have uncovered both similarities and differences between OR and TAAR gene expression?some of which raise fundamental questions about current models of OR choice. Reconciling these differences and shedding light on TAAR expression are both critical to our understanding of gene choice mechanisms in olfaction. The goal of this proposal is to characterize for the first time genetic elements that are involved in monogenic expression of TAAR genes. I have identified two well-conserved, putative regulatory elements in the TAAR gene cluster. I propose to use advanced mouse genetic approaches to test whether and how these elements contribute to TAAR gene choice. Specific Aim 1 will use transgenesis and gene targeting via CRISPR/Cas9 to test whether one or both of these elements is sufficient to drive singular expression in specific populations of olfactory sensory neurons that normally express TAARs. Specific Aim 2 will use genome editing via CRISPR/Cas9 to determine if one or both of these elements is necessary for singular expression of TAAR genes. These experiments provide necessary first steps towards characterizing the similarities and differences between OR and TAAR gene choice mechanisms. Such comparisons will provide insight into the basic mechanisms of monoallelic gene expression and cell type diversity in the mammalian nervous system.